Fluorescent dyes are known to be particularly suitable for biological applications in which a highly sensitive detection reagent is desirable. Fluorescent dyes are used to impart both visible color and fluorescence to other materials.
The dyes of this invention are fluorine-substituted analogs of xanthene-based dyes that are typically fluorescein or rhodol derivatives. Fluoresceins and rhodols are known to strongly absorb visible light and, in most cases, to be highly fluorescent. Polychlorinated, polybrominated and polyiodinated analogs of fluorescein dyes are known to shift the spectrum to longer-wavelengths than the unsubstituted dyes. Polybrominated and polyiodinated analogs of fluorescein dyes have much lower fluorescence yields, higher phosphorescence yields and are effective photosensitizers of certain chemical reactions. The preparation and use of reactive rhodol dyes and their conjugates was disclosed in U.S. Pat. Nos. 5,227,487 (1993) and 5,442,045 (1995) to Haugland et al. (each incorporated by reference). Both fluorescein and rhodol are considered "xanthene" dyes. That is, they are characterized by structural similarity to, or derivation from, xanthene. ##STR1##
"Fluorescein" dyes include derivatives of 3H-xanthen-6-ol-3-one that are substituted at the 9-position by a 2-carboxyphenyl group, while "rhodol dyes" include derivatives of 6-amino-3H-xanthen-3-one that are substituted at the 9-position by a 2-carboxyphenyl group. ##STR2##
When substituted at the 1-position by a derivative capable of forming a 5- or 6-membered lactone or lactam ring alternative structures for fluorescein and rhodol dyes are possible: ##STR3##
Despite their widespread use in biological assays, fluorescein-based dyes have certain well-known deficiencies. In particular, it is widely recognized that fluorescein conjugates are unstable with respect to the intense illumination produced in most fluorescence instrumentation. The result is irreversible photobleaching, which typically results in a rapid decrease in fluorescent signal. In a fluorescence microscope the amount of irreversible bleaching may reach essentially 100% in less than one minute. This susceptibility to photobleaching considerably reduces the utility of fluorescein fluorophores for quantitative assays. A second problem of fluorescein is that its pK.sub.a (acid dissociation constant) is approximately 6.4 in water (see FIG. 3), making its absorbance and fluorescence properties quite pH dependent in the physiological pH range, so much so that certain fluoresceins are utilized as fluorescent pH indicators (for example BCECF, Rink et al. J. CELL BIOL. 95, 189 (1982)). This pH sensitivity also reduces the utility of fluoresceins for quantitative assays (See FIG. 3). A third difficulty that is recognized when using fluorescein dyes is the tendency of fluorescein conjugates to exhibit fluorescence that is highly quenched relative to that of the free fluorophore. This typically quickly leads to protein conjugates of fluorescein exhibiting less fluorescence even when more dye molecules are conjugated to the protein (see FIG. 1), reducing the sensitivity that is possible for assays using fluorescein conjugates. A highly desirable class of fluorophores would retain the favorable characteristics of fluorescein, including generally high absorbance and fluorescence of fluorescein, the ability to be excited using the 488-nm line of the argon-ion laser, and the ability to use standard optical filters for fluorescein that are already available for most fluorescence microscopes, while simultaneously possessing characteristics that improve on the deficiencies of fluorescein.
Fluorination of fluorescein-like dyes simultaneously improves all three of these known deficiencies of fluorescein dyes, while generally retaining the other desirable spectral properties of fluorescein-like dyes. In particular, fluorinated analogs of fluorescein are typically more photostable than the corresponding nonfluorinated analogs. In addition, the conjugates of fluorinated fluorescein dyes typically exhibit higher fluorescence than those of nonfluorinated dyes. In some cases the fluorescence of the protein conjugates of the fluorinated dyes is not quenched at degrees of substitution that strongly quench the fluorescence of conjugates of the corresponding nonfluorinated dyes (for example, see FIG. 1). The pK.sub.a of the fluorinated dyes is significantly lower than that of the nonfluorinated analogs (&gt;1.5 pH units on the otherwise unsubstituted dyes, see FIG. 3). Furthermore, we found that fluorination of fluoresceins at the 2'- and 7'-positions unexpectedly had virtually no effect on either the absorption spectrum, the fluorescence spectrum or the quantum yield of the dye relative to the nonfluorinated versions, and additionally, the conjugates of these fluorinated fluoresceins tend to be more fluorescent than the nonfluorinated versions (for example, see FIG. 1) making these dyes preferred substitutes for fluoresceins. All other reported substitutions at the 2'- and 7'-positions of fluorescein, including halogenation with Cl, Br or I, have been reported to shift the spectra of the dye toward longer wavelengths.
Additionally, fluorescein analogs that are polyfluorinated on the phenyl substituent that is typically present in most fluorescein and rhodol dyes are subject to nucleophilic displacement reactions, providing a novel route to reactive derivatives and conjugates. Certain types of the fluorinated xanthenes unexpectedly give products that are well-retained in live biological cells. This reactivity is not observed with the nonfluorinated xanthenes.
In addition to the fluorinated dyes of the invention, we hereby also provided a means to prepare derivatives of fluorescein that are substituted at the 1'- and/or 8'-positions; molecules that do not appear to have been previously described.
Analogs of fluorescein dyes with fluorine atoms substituted onto the xanthene portion of the dye have been generically included as claimed embodiments in various patents describe, typically by claiming "halogens" as permitted substituents on the dye. However these patents do not describe either the highly advantageous properties of these fluorinated fluorophores and their conjugates, and typically require other structural limitations not required by the present invention. In particular these patents do not describe the lack of spectral shift and enhanced photostability that typifies several of the dyes of the current invention. Also, these patents do not require that the dye be substituted by at least one fluorine atom. Furthermore, these patents fail to describe methods that are suitable for synthesis of the dyes the invention, as the unique chemistry of fluorine in organic chemistry does not permit utilization of the methods that are typically used to prepare chlorine, bromine or iodine substituted fluorescein derivatives. While halogenated rhodol dyes have been described previously (U.S. Pat. Nos. 5,227,487 (1993) and 5,442,045 (1995) to Haugland et al., the properties and syntheses of fluorinated analogs of rhodol dyes have not previously been described. In particular, efficient methods for preparing the requisite fluororesorcinols and fluoroaminophenols have not previously been described. It was found that a more facile method of preparing these intermediates was required in order to efficiently prepare the wide variety of dyes described herein.
The preparation and use of fluorescein dyes and conjugates of fluorescein dyes is already well-known in the art, as described in U.S. Pat. Nos. 4,213,904 to Haugland et al. (1980), 3,996,345 to Ulman et al. (1976), 4,199,559 to Ullman et al. (1980)), 5,352,803 to Mattingly (1993), 4,420,568 to Wang et al. (1983), 5,332,666 to Prober et al. (1994), 4,997,928 to Hobbs (1991), 4,439,356 to Khanna et al. (1984), 4,519,251 to Kirkemo et al. (1985), 4,609,740 to Rotman (1986), 4,318,846 to Khanna et al. (1982), and 5,451,343 to Neckers et al. (1995), all of which are incorporated by reference, and International Publication No. WO 94/05688 by Menchen et al. (1994).
We have demonstrated the use of the fluorinated dyes for the preparation of a wide variety of reactive derivatives that utilize chemistry mostly analogous to that used to prepare known reactive fluorescein and rhodol dyes. We have also demonstrated a heretofor unknown reactivity toward nucleophilic displacement possessed by fluorinated dye derivatives that are fluorinated on the 9-aryl portion of the dyes. We have further demonstrated that the improved properties of the fluorinated dyes are retained in their conjugates and that these conjugates have properties that are typically superior to those prepared from the corresponding nonfluorinated dyes. The dyes of the invention are suitable for substitution for fluoresceins and rhodols in all their known applications except those requiring sensitivity to pH changes near pH 7.
For the practical preparation of the fluorinated dyes it was necessary to develop a novel general synthesis of fluorinated resorcinol and aminophenol derivatives that would yield isomer-free products in good yield. Previous methods give low yields of products that usually consist of difficult-to-separate isomer mixtures and require the use of generally obnoxious fluorinating agents. Our novel method uses generally available precursors and standard laboratory reagents and equipment to reproducibly produce these essential precursors in relatively large quantities.
It is an object of this invention to provide novel and advantageous fluorinated fluorescein and rhodol dyes suitable for use as tracers, labels or synthetic intermediates. It is an additional object of this invention to provide reactive derivatives of fluorinated fluorescein and rhodol dyes useful for the preparation of fluorescent dye-conjugates. It is yet another object of this invention to provide usefully labeled dye-conjugates of fluorinated fluorescein and rhodol dyes that possess the novel and advantageous properties of the novel dyes. It is a further object of the invention to provide versions of fluorinated fluorophores that are useful for detecting enzymes, particularly within live cells and cellular organelles. It is also an object of the invention to provide a novel synthesis of fluorinated resorcinols and aminophenols, greatly simplifying the preparation of the fluorinated fluorescein and rhodol dyes of the invention.